Submitted to: Applied and Environmental Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 2/18/2004
Publication Date: 6/1/2004
Citation: Schweigkofler, W., O Donnell, K., Garbelotto, M. 2004. Detection and quantification of airborne conidia of Fusarium circinatum, the causal agent of pine pitch canker, from two California sites by using a novel real time-PCR approach combined with a simple spore trapping method. Applied and Environmental Microbiology. 70(6):3512-3520. Interpretive Summary: Monterey pine is the tree most widely planted for timber production worldwide. Pine pitch canker disease, caused by the fungus Fusarium circinatum, is a major threat to the commercial production of Monterey pine. Pine pitch canker was first detected in the Southeastern U.S., but it has rapidly spread to California, Mexico, South Africa, Japan and Chile. Long-range spread of the disease appears to be connected to the movement of infected plant material for commercial use. To address this problem, we developed a rapid, novel molecular diagnostic test to detect the presence of this devastating pine pathogen well before symptoms appear on trees, or on material transported commercially to prevent further spread of the disease. The accuracy of this novel test for the active surveillance of the pine pathogen was validated through both field and laboratory experiments. These results will benefit quarantine officials and plant disease specialists at the state and federal levels by providing novel molecular tools to help prevent the further spread of this economically important disease.
Technical Abstract: Monterey pine (Pinus radiata D. Don), a species native to California and Northern Mexico, is the tree most widely planted for timber production worldwide. Plantations of this species cover several million hectares mainly in New Zealand, Australia, South Africa, Chile and Spain. Pine pitch canker, caused by the ascomycete fungus Fusarium circinatum, is a major threat to commercial production of Monterey pine. We developed a novel trapping approach using filter paper in combination with a rapid molecular method to detect the presence of inoculum well before symptoms appear on trees, or on material transported commercially. The test is based on the F. circinatum specific primer pair CIRC1A-CIRC4A, which amplify a 360 bp DNA fragment in the intergenic spacer region (IGS) of the nuclear ribosomal operon. Real-time PCR was used to calculate the starting copy number of target sequences (i.e. = fungal spores) present in each reaction by comparing the threshold cycle (Ct) of unknown spore samples to the Ct values of standards with known amounts of F. circinatum DNA and spores, respectively. The filter paper method allows for prolonged and more sensitive spore sampling in the field when compared to traditional traps using Petri dishes filled with selective medium. In addition, other genetic information such as the mating type of F. circinatum obtained from spore traps can be determined for spores collected en masse. All samples from California analyzed to date belonged to the MAT-1 mating type.